Both types I and II diabetes mellitus result from abnormal function or loss of insulin-secreting beta cells in pancreatic islets. Beta cell membranes govern ionic conductances, exocytosis, and antigenic properties, and complex lipid components affect membrane functions. Characterization of islet lipids is difficult because islets are obtainable in only small quantities. Conventional methods of complex lipid analysis involve multiple chromatographic, digestion, and derivatization steps that each involve analyte losses, and FAB/MS is insufficiently sensitive to permit analysis of complex lipids in islet subcellular membranes. The great sensitivity of ESI/MS combined with structural information provided by collisionally activated dissociation and tandem mass spectrometry circumvents these difficulties. We have developed ESI/MS/MS methods that facilitate identification of small quantities of glycerophosphocholine lipids and of sulfatide species. ESI/MS has been used to identify a ll major phospholipid species in islet secretory granules and plasma membranes, to identify islet sulfatide species, and to examine stimulus-induced changes in amounts of islet phospholipid species. Characterization of islet lipids has led to the discovery of a new lipid metabolizing enzyme, which is now designated iPLA2. We have overexpressed this enzyme in Sf9 cells and are characterizing its post-translational modifications by HPLC/ESI/MS/MS analyses of tryptic digetsts of the recombinant protein.